In our hospital with an established infection control program designed to contain transmission from symptomatic CDI patients, asymptomatic carriers appear to have played an important role in transmission. Identification and isolation of carriers may be necessary to further reduce transmission of C. difficile in such settings.

bRecurrent Clostridium difficile infection (CDI) occurs in up to 35% of patients. Recurrences can be due to either relapse with the same strain or reinfection with another strain. In this study, multilocus variable-number tandem-repeat analysis (MLVA) was performed on C. difficile isolates from patients with recurrent CDI to distinguish relapse from reinfection. In addition, univariate and multivariate analyses were performed to identify risk factors associated with relapse. Among patients with a single recurrence, relapse due to the original infecting strain was more prevalent than reinfection and the interval between episodes was shorter than among patients who had reinfections. Among patients with >1 recurrence, equal distributions of relapse and reinfection or a combination of the two episode types were observed. Initial infection with the BI/NAP1/027 epidemic clone was found to be a significant risk factor for relapse. This finding may have important implications for patient therapy. Classification of recurrent CDI episodes by MLVA can be utilized to make informed patient care decisions and to accurately define new CDI cases for infection control and reimbursement purposes. One of the most problematic aspects of Clostridium difficile infections (CDI) is the propensity of recurrence in 15% to 35% of patients who initially respond to antimicrobial therapy. In addition, recurrent CDI is difficult to treat and contributes to significant morbidity and mortality and increased health care expenditures (10,11,27).The molecular epidemiology of CDI has changed since 2000 with the global spread of an epidemic clone, designated BI/NAP1/ 027 by restriction endonuclease analysis (REA), pulsed-field gel electrophoresis, and PCR ribotyping. However, recent studies have raised doubts regarding the role of BI/NAP1/027 in increased CDI incidence, severity, and recurrence rates (4, 21).Recurrent CDI can caused by either relapse due to the original infecting strain or reinfection with a new strain. Previous studies have demonstrated that continued non-CDI antibiotic treatment and a failed immune response to C. difficile toxins A and B are risk factors for recurrent CDI (15,20,25). Most recently, lower cure rates and higher rates of recurrence due to BI/NAP1/027 infection were reported in phase 3 clinical trials of fidaxomicin (26). Recent estimates suggest that 65% to 88% of recurrent CDI is attributable to relapse with the original infecting strain (2, 3, 12). However, some of the molecular typing methods used in these studies such as PCR-ribotyping and random amplification of polymorphic DNAs (RAPD) lack sufficient discriminatory power and may have misclassified reinfections as relapses. In a study using restriction endonuclease analysis (REA), 83.3% of recurrences were due to relapse (8). In this study, multilocus variable-number tandemrepeat analysis (MLVA), a highly discriminatory C. difficile genotyping method, was used to define relapse in patients with recurrent CDI and to identify risk factors associated with relapse. ...

bPrevious studies suggested that 7 to 15% of healthy adults are colonized with toxigenic Clostridium difficile. To investigate the epidemiology, genetic diversity, and duration of C. difficile colonization in asymptomatic persons, we recruited healthy adults from the general population in Allegheny County, Pennsylvania. Participants provided epidemiological and dietary intake data and submitted stool specimens. The presence of C. difficile in stool specimens was determined by anaerobic culture. Stool specimens yielding C. difficile underwent nucleic acid testing of the tcdA gene segment with a commercial assay; tcdC genotyping was performed on C. difficile isolates. Subjects positive for C. difficile by toxigenic anaerobic culture were asked to submit additional specimens. One hundred six (81%) of 130 subjects submitted specimens, and 7 (6.6%) of those subjects were colonized with C. difficile. Seven distinct tcdC genotypes were observed among the 7 C. difficile-colonized individuals, including tcdC genotype 20, which has been found in uncooked ground pork in this region. Two (33%) out of 6 C. difficile-colonized subjects who submitted additional specimens tested positive for identical C. difficile strains on successive occasions, 1 month apart. The prevalence of C. difficile carriage in this healthy cohort is concordant with prior estimates. C. difficile-colonized individuals may be important reservoirs for C. difficile and may falsely test positive for infections due to C. difficile when evaluated for community-acquired diarrhea caused by other enteric pathogens.C lostridium difficile infections (CDIs) have shown dramatic increases in incidence and morbidity during the past decade (1). While C. difficile is well established as a health careassociated pathogen, incidence estimates for community-acquired CDIs vary between 6 and 30% (2-4). The source of C. difficile in community-acquired cases is unclear. Prevalence estimates for asymptomatic C. difficile colonization range from 7 to 15% among healthy, non-health care workers outside the United States to 1 to 4% among health care workers in the United States and abroad (5-9). Individuals colonized with C. difficile could represent a potential reservoir of strains imported into hospitals, as well as a source of false-positive clinical test results for CDIs among patients with community-acquired diarrheal illness, especially norovirus and Clostridium perfringens. We studied the prevalence, genotypic distribution, and duration of C. difficile carriage among healthy adults in Allegheny County, Pennsylvania, and evaluated potential risk factors for asymptomatic colonization. We also examined the relative abundance of C. difficile and the sensitivity of a commercial nucleic acid test for detection of C. difficile in the stool of healthy colonized individuals. MATERIALS AND METHODSHealthy adult residents (Ն18 years of age) in Allegheny County, Pennsylvania, were recruited for participation via print and electronic advertisements in September 2012 through April 2013. Ind...

Increased incidence of infections due to Klebsiella pneumoniae carbapenemase (KPC)-producing Klebsiella pneumoniae (KPC-Kp) was noted among patients undergoing endoscopic retrograde cholangiopancreatography (ERCP) at a single hospital. An epidemiologic investigation identified KPC-Kp and non-KPC-producing, extended-spectrum β-lactamase (ESBL)-producing Kp in cultures from 2 endoscopes. Genotyping was performed on patient and endoscope isolates to characterize the microbial genomics of the outbreak. Genetic similarity of 51 Kp isolates from 37 patients and 3 endoscopes was assessed by pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST). Five patient and 2 endoscope isolates underwent whole genome sequencing (WGS). Two KPC-encoding plasmids were characterized by single molecule, real-time sequencing. Plasmid diversity was assessed by endonuclease digestion. Genomic and epidemiologic data were used in conjunction to investigate the outbreak source. Two clusters of Kp patient isolates were genetically related to endoscope isolates by PFGE. A subset of patient isolates were collected post-ERCP, suggesting ERCP endoscopes as a possible source. A phylogeny of 7 Kp genomes from patient and endoscope isolates supported ERCP as a potential source of transmission. Differences in gene content defined 5 ST258 subclades and identified 2 of the subclades as outbreak-associated. A novel KPC-encoding plasmid, pKp28 helped define and track one endoscope-associated ST258 subclade. WGS demonstrated high genetic relatedness of patient and ERCP endoscope isolates suggesting ERCP-associated transmission of ST258 KPC-Kp. Gene and plasmid content discriminated the outbreak from endemic ST258 populations and assisted with the molecular epidemiologic investigation of an extended KPC-Kp outbreak.

g Clostridium difficile is the most commonly identified pathogen among health care-associated infections in the United States. There is a need for accurate and low-cost typing tools that produce comparable data across studies (i.e., portable data) to help characterize isolates during epidemiologic investigations of C. difficile outbreaks and sporadic cases of disease. The most popular C. difficile-typing technique is PCR ribotyping, and we previously developed methods using fluorescent PCR primers and amplicon sizing on a Sanger-style sequencer to generate fluorescent PCR ribotyping data. This technique has been used to characterize tens of thousands of C. difficile isolates from cases of disease. Here, we present validation of a protocol for the cost-effective generation of fluorescent PCR ribotyping data. A key component of this protocol is the ability to accurately identify PCR ribotypes against an online database (http://walklab.rcg.montana.edu) at no cost. We present results from a blinded multicenter study to address data portability across four different laboratories and three different sequencing centers. Our standardized protocol and centralized database for typing of C. difficile pathogens will increase comparability between studies so that important epidemiologic linkages between cases of disease and patterns of emergence can be rapidly identified. C lostridium difficile infection (CDI) has emerged as the most frequently encountered nosocomial infection in the UnitedStates (1). The clinical manifestations of CDI range from acute, self-limiting diarrhea to fulminant and sometimes fatal colitis (2). Over the past decade, there has been a doubling of CDI-related discharge diagnoses and a 10-fold increase in CDI-attributable mortality in the United States (3). Because of its clinical importance, a number of C. difficile-typing techniques, including pulsed-field gel electrophoresis (PFGE), restriction endonuclease analysis (REA) typing, and PCR ribotyping, have been implemented to differentiate between C. difficile strains and address important epidemiologic questions.PCR ribotyping is the most commonly cited method for typing C. difficile isolates. This technique quantifies the differences in length between 16S rRNA and 23S rRNA encoding genes at approximately 11 rRNA-encoding operons around the C. difficile genome (4). Like other popular typing techniques (REA and PFGE), PCR ribotyping is gel based, and the data are not easily portable between laboratories. Sequence-based methods, like multilocus sequence type (MLST) and genome sequencing, provide highly portable data but are expensive in comparison to gelbased methods. A significant improvement on traditional PCR ribotyping came with the use of a fluorescently labeled PCR primer and sizing of the resulting amplicons using a Sanger-style sequencer (i.e., fluorescent PCR ribotyping) (5). We developed a similar approach and have applied the method to C. difficile isolates from cases of disease and those that circulate in the community (6-11).Fluorescent PC...